1. Field of the Invention
The invention relates to a method of detecting viable cells in a sample, in particular, to a method of accurately detecting viable cells, the method facilitating discrimination between a viable cell and an inclusion, such as a dust particle, by staining the sample using a fluorescent dye and a quenching dye.
2. Description of the Related Art
In the fields of medicines, agricultural chemicals, and food health control, and in the research fields of medical science, pharmacy, and biology, the detection and measurement of viable cells in a sample are often conducted for quality control and assessing safety and the effect of drugs. To detect and measure viable cells quickly and readily, detection methods have been proposed in which the viable cells are stained by using a staining reagent.
For example, Japanese Patent No. 2979383 discloses a method of quantification of live and dead microbes using fluorescent dyes of fluorescein diacetate and propidium iodide. In the method, the microbes are doubly stained by these dyes and illuminated by excitation light. Detecting fluorescent light at a specific wavelength emitted from the live cells stained with fluorescein diacetate and fluorescent light at a specific wavelength emitted from the dead cells stained with propidium iodide, the numbers of live and dead cells are quantified based on the intensity of the emitted fluorescent light at the respective wavelengths. The method of Japanese Patent No. 2979383, however, has a drawback in that the fluorescein diacetate is vulnerable to dissociation and staining of other inclusions than viable microbes, inhibiting accurate quantification of viable cells in the case of samples containing live microbes, dead microbes, and other inclusions.
Japanese Unexamined Patent Application Publication No. 2003-169695 discloses a method of quantification of microbes to count live and dead cells in two counting steps and then comparing the results in the two steps. That is, in the first step of the method, the entire microbe sample is stained with a fluorescence reagent that allows only dead cells to emit fluorescent light, and fluorescent dead cells are counted. In the second step, after sterilization of the entire sample, the entire sample is stained again with the same fluorescent reagent and the fluorescent dead cells are counted. By subtracting the intensity of the first measurement from the intensity of the second measurement, the numbers of live and dead cells in the initial sample are quantified. However, the method of Japanese Unexamined Patent Application Publication No. 2003-169695 also involves disadvantages. In addition to the need for a complicated process of sterilization, the conditions of the sterilization affect the measured result and must be closely examined.
Japanese Unexamined Patent Application Publication No. H10-99096 discloses a method of measuring a number of viable cells and/or a viability ratio. The method measures an intensity of fluorescent light emitted from a sample treated with a nucleic acid fluorescent dye that allows only dead cells to be stained, and measures an intensity of fluorescent light emitted from a sample treated with the nucleic acid fluorescent dye and subjected to a treatment to disrupt the cell membrane. The two intensities are subtracted from each other to determine the number of viable cells in the original sample, or the two intensities are divided to determine the viability ratio.
The method of Japanese Unexamined Patent Application Publication No. H10-99096 also involves problems. The treatment to disrupt the cell membrane is troublesome, and the conditions of the treatment must be closely checked. The objects of the measurement are undercounted since the cells having a cell wall cannot be measured. Also, a typical dye used, Trypan blue, is a toxic substance requiring careful handling.
Japanese Unexamined Patent Application Publication No. 2002-34594 discloses a method of detecting viable cells that includes a process of detection or measurement of a dye or fluorescence in a cell sample having a dye or a fluorescent enzyme substrate added, wherein the detection or the measurement is carried out using an absorber that is impermeable through a cell membrane and absorbs the dye or the light emission from the fluorescent enzyme substrate. The fluorescent enzyme substrate is a compound selected from the group consisting of 5-carboxyfluorescein diacetate acetoxymethyl ester, 5-(6-) carboxyfluorescein diacetate, 2′,7′-bis-(2-carboxyethyl)-5-(6-) carboxyfluorescein acetoxymethyl ester, 5-(6-) sulfofluorescein diacetate, fluorescein diacetate, calcein acetoxymethyl ester, 5-chloromethyl fluorescein diacetate, 5-(6-) carboxyfluorescein diacetate succinimidyl ester, and fluorescein-5-carbonylazido diacetate. The dye is a compound selected from the group consisting of acridine orange, bis-benzimidofluorochrome trihydrochlorate, 4′,6′-diamino-2-phenyl indole, fluorescent nucleic acid stains SYTO®9 (green), SYTO®10(green), SYTO®11(green), SYTO®12(green), SYTO®13(green), SYTO®14(green), SYTO®15(green), SYTO®16(green), SYTO®17(red), SYTO®20(green), SYTO®21(green), SYTO®22(green), SYTO®23(green), SYTO®24(green), SYTO®25 (green) (SYTO is a registered trademark of Molecular Probes, Inc., a Subsidiary of Invitrogen Corp.), hexidium iodide, and dihydroethidium. The absorber is a compound selected from the group consisting of cytochrome C, hemoglobin, and blue dextran.
In a paper published in the Journal of Immunological Methods, 100 (1987), pp. 261-267, Y. Hansson, et al. disclose a method of quantifying viable cells. In the method, the sample is doubly stained using carboxyfluorescein diacetate (CFDA), which is a reagent for viable cells to emit fluorescent light; and hemoglobin, which quenches the fluorescence of unreacted CFDA emitted from the viable cells. The stained sample is illuminated with an excitation light and the emitted fluorescence from the viable cells stained by the CFDA is detected by a photomultiplier connected to a fluorescent microscope, to determine the number of viable cells from the detected intensity of the fluorescence.
Problems in the methods of Japanese Unexamined Patent Application Publication No. 2002-34594 and the Y. Hansson, et al. publication include that the absorber of the emitted light from the dye or fluorescent enzyme substrate is a protein such as cytochrome C, hemoglobin or the like. Such a protein needs cold storage and it is therefore difficult to stably maintain its quality. Moreover, blue dextran is expensive.
U.S. Pat. No. 6,459,805 discloses a method of quantifying a relative amount of viable cells by staining a sample containing the viable cells with two types of dyes and measuring the intensity of fluorescence from the sample. The first of the two types of dyes is a fluorescent dye that is accumulated in viable cells only. The second is a dye that quenches the fluorescence of the fluorescent dye and is permeable to dead cells but excluded by viable cells. The disclosed combinations of the dyes include a combination of a fluorescent dye of fluorescein diacetate and a quenching dye of eosin Y, as well as a combination of a fluorescent dye of calcein-AM and a quenching dye of trypan blue. The method of U.S. Pat. No. 6,459,805 suffers from a problem in fast quantification because a relatively long time (about 30 min) is required for staining the sample with the fluorescent dye. In addition, in one of the examples, the quenching dye eosin Y does not absorb the fluorescent light of the carboxyfluorescein diacetate in the wavelength range between 550 nm to 650 nm, so eosin Y cannot quench the fluorescence of the fluorescein diacetate satisfactorily.